T1DM results from the immune-mediated destruction of the body's only cells capable of physiologically regulated insulin secretion, the pancreatic beta cells. Beta cells comprise an estimated 2% of the pancreatic cell number and are grouped together into cell clusters with alpha cells (making glucagon), delta cells (making somatostatin), F cells (making pancreatic polypeptide), and other more rare cells into "mini-organs" called the islets of Langerhans. While the isolated destruction of islet alpha, delta, or F cells has not been described in the clinic, isolated T lymphocyte-mediated beta cell destruction is quite common as it is thought to underlie T1DM (and animal models of T1DM). Pancreata from animal models or from patients with newly diagnosed T1DM reveal "islet remnants", i.e. small islets with few or no beta cells while the other islet cell types persist. Over time however, clinical evidence suggests that patients with T1DM also lose much alpha cell function suggesting that it too is dependent upon normal beta cell function. The islet as "mini-organ" concept is apt for at least 2 other reasons: (1) while the cellular organization is different for different species, the various islet cell types are nevertheless organized in a very orchestrated way, and (2) islets consume more pancreatic blood flow (about 20%) than their small mass would suggest. Thus islets are important if incompletely understood structures with unique anatomical, physiological, and immunological features; and they represent an Achilles' heel for individuals destined to develop T1DM. This project has multiple components, to: (1) characterize isolated islet structure, function, and quality, (2) test, using a non-human primate islet transplant model, important pre-clinical questions, (3) perform human clinical islet transplants using carefully planned protocols, (4) develop clinical assays for characterizing islet function pre- and post-transplant, (5) develop a renewable islet source, and (6) test novel ways of preventing islet allograft rejection following transplant. Beginning in 7/99, in collaboration with the Clinical Center's Department of Transfusion Medicine/Cell Processing Unit and Drs. Ricordi and Kenyon (University of Miami's Diabetes Research Institute), islets have been isolated from both human and non-human primate pancreata. Many of these islets have been shared with various intra- and extra-mural collaborators pursuing shared goals as enumerated above. To enable these isolations, we established two separate laboratories, one for human glands and one for animals, and we trained a team of technicians. We perform the standard in vitro islet function assays (islet insulin release in low- and high-glucose media, viability assays, and more recently islet peri-fusion assays). In addition, we've subjected some islet preps to the gold-standard islet function assay (i.e. transplanting the islets into diabetic NOD-scid mice), initially with our collaborators at Vanderbilt University (Dr. Alvin Powers) but we are now establishing the assay intramurally. We have also performed new in vitro islet function assays and through that research made several novel observations including: a) RNA expression "microarray" data demonstrating islet gene expression patterns, in particular highly regulated TGF-beta gene expression, b) a never-before appreciated insulin RNA splice form translated into protein more efficiently the native splice form, and c) data demonstrating that islets produce the hormone "resistin" here-to-fore thought to be expressed only in fat. Each of these observations has now been published in a peer-reviewed journal. Further, several collaborators are attempting various techniques to culture islets in vitro so as to increase islet function and/or number. For instance, this year and in collaboration with Dr. Ricarrdo Perfetti, we published that treating islets in vitro with the hormone GLP-1 improves islet function and decreases islet death. Our non-human primate experiments also yielded several important observations including: a) diabetes can be effectively and more safely induced (compared with the more typically employed surgical pancreatectomy approach) via intra-arterial infusion of a beta cell toxin call streptozotocin (publication in press), b) the immunohistological and metabolic consequences of islets transplanted into the liver, and c) novel calcineurin phosphatase inhibitor free immunomodulatory regimens can support islet allograft survival. All these studies are designed to support NIH islet transplant clinical trials. Of course, the most immediately relevant product of our research is the knowledge gained from our clinical islet transplant experience including 6 patients with long-standing and "brittle" T1DM. Detailed metabolic testing on the patients found that patients had basically normal insulin sensitivity, but that even the insulin independent patients had marginal islet function and imperfect glycemia control. Due to our belief that the factors limiting islet transplantation (primarily the inadequate donor islet supply, and imperfect immunosuppressive regimens) were not being effectively and most safely addressed by the solitary islet transplant protocol, we suspended patient accrual, but have published several observations from our experience, with several other publications in press. For instance, we have data suggesting that pancreas transplantation and the subsequent post-transplant anti-rejection therapy when performed for individuals with diabetes and preserved kidney function, may result in worse survival than that observed in patients receiving more traditional diabetes therapies. We are completing preparations for a combined kidney-islet transplant clinical protocol to test novel immunosuppressive regimens and islet transplantation techniques in patients with T1DM induced renal failure and therefore who already require a kidney allograft and chronic immunosuppression. Using this protocol, we hope to test whether pancreatic islet function can be restored for individuals with long-standing T1DM, and if so, to characterize factors to promote that pancreatic islet function.